Human NF-kappaB (NF-KB) transcription factors regulate the expression of large number of genes involved in diverse biological activities. A sub-class of IKB proteins, known as prototypical IKB (IKBalpha, IKBbeta and iKBepsilon), inhibits NF-KB transcriptional activity by forming stable complexes with NF-KB dimers. These inhibited complexes define a paradigm of cell signaling where signal induced NF-KB activation is regulated by the degradation of IKB. The prototypical IKB proteins undergo rapid turnover in resting cell when present in the free state, compared to when present as complexes with NF-KB. Recent observations indicate that the prototypical IKBs are partly unfolded, which may stabilize upon binding NF-KB. Interestingly, critical segments of NF-KB that are involved in binding IKB are also unfolded in their free states. We hypothesize that stability of IKB proteins regulates their turn over, NF-KB recognition and the lifetime of IKB/NF-KB complexes in quiescent cells. A separate class of IKB proteins, represented by Bcl3 and IKBzeta, carry out different functions. It is likely that functional differences between these two classes of 1KB proteins may correlate with their differential stability. The focus of this proposal is to understand the mechanism of proteasome-mediated degradation of IKB and how their stability relates to their degradation. We have observed structural differences between prototypical IKB proteins such as the presence of a disordered insert in IKBbeta. We will test if IKBalpha and IKBbeta display differential specificity towards specific NF-KB dimers, and how structural differences between IKBalpha and IKBbeta play a role in such specificity-defining mechanisms. Finally, we will elucidate new structures of IKB/NF-KB complexes, which may provide insights into their biological functions. We will use X-ray crystallography, biochemistry and cell-based experiments to test our hypotheses.